Antibody molecules, including IgG, are now indispensable tools in the fields of research, treatment, and diagnosis due to their binding properties to antigen molecules. In general, antibody molecules are multi-domain glycoproteins having a large molecular weight, and there are a plurality of disulfide bonds (hereinafter, sometimes simply referred to as S—S bonds) in the molecule, so it is necessary to express the proteins using animal cells. Production of proteins using animal cells, however, is generally more expensive than production using bacteria, and recombinant antibody molecules often have poor expression, so there is a problem of low productivity.
In order to supplement for the foregoing problems in the production of antibody molecules, use of a fragment antibody having a small molecular weight has been devised. Fragment antibodies have merits such as high tissue invasiveness.
A representative fragment antibody may be, for example, Fab consisting of variable domains (VH region and VL region) and constant domains (CH1 region and CL region). However, there is a problem of high production costs since Fab is generally obtained by purifying a full-length antibody, subjecting the purified full-length antibody to an enzymatic treatment, and subjecting the enzymatically treated antibody to further purification. In addition, a single-chain Fv (hereinafter, sometimes simply referred to as scFv) in which a C-terminus of the VH region or VL region and the other N-terminus are linked by a long and flexible peptide linker is known as another fragment antibody (Non-Patent Literature 1). However, aggregation of scFvs sometimes occurred due to the presence of the long and flexible peptide linker. In addition, there was also a problem that the activity as an antibody is lowered or destabilized.
Then, in order to solve the problems of the conventional fragment antibodies, the present inventors have developed a novel fragment antibody (hereinafter, sometimes simply referred to as Fv-clasp first generation or Fv-clasp (v1)) in which the C-termini of the VH region and VL region are linked through a dimer of SARAH domains of human mammalian sterile 20-like kinase 1 (Mst1) which forms an antiparallel coiled coil (Non-Patent Literature 2).
This Fv-clasp (v1) is a fragment antibody consisting of a complex of (a) a peptide in which the N-terminus of the SARAH domain of human Mst1 is linked to the C-terminus of a heavy chain domain (VH region) of the antibody and in which an amino acid residue at position 35 from the N-terminus of the SARAH domain of the human Mst1 is mutated to cysteine, and (b) a peptide in which the N-terminus of the SARAH domain of human Mst1 is linked to the C-terminus of a light chain domain (VL region) of the antibody and in which an amino acid residue at position 24 from the N-terminus of the SARAH domain of the human Mst1 is mutated to cysteine, in which (c) two SARAH domains are linked by a disulfide bond between the mutated cysteines.